title: Development of multiplex PCR assays based on the 16S-23S rRNA internal transcribed spacer for the detection of clinically relevant nontuberculous mycobacteria.
abstract: OBJECTIVE
  To accelerate the identification and differentiation of clinically relevant nontuberculous mycobacteria (NTM) with two sets of multiplex PCR (mPCR) targeting the 16S-23S rRNA internal transcribed spacer (ITS) region for timely patient management.
  
  
  RESULTS
  Two mPCR assays were developed: Slow-Growers (SG) mPCR was used for the detection of slow-growing mycobacteria, which included Mycobacterium avium complex, Mycobacterium kansasii, Mycobacterium gordonae and Mycobacterium xenopi whilst the other mPCR assay labelled as Fast-Growers (FG) mPCR was used for the detection of Mycobacterium fortuitum complex, Mycobacterium abscessus and Mycobacterium chelonae. In these assays, a common forward primer based on a conserved section of the 16S rRNA region was used in conjunction with species-specific reverse primers. The mPCRs were tested against 247 clinical mycobacterial isolates and demonstrated 100% specificity and sensitivity. Identification of the mycobacterial species was also validated by DNA sequencing of the 16S-23S ITS region and when further confirmation was needed, hsp65 sequencing was performed.
  
  
  CONCLUSIONS
  The mPCR assays could be a potentially useful diagnostic tool for the rapid and accurate identification of clinically relevant NTM.
  
  
  CONCLUSIONS
  In this study, we looked at the frequency of hospital isolated NTM over the last 5 years (2005-2010), and an mPCR targeting the ITS region was developed for NTM species that appeared to be more prevalent in the context of Singapore.
authors.affiliation: National University Hospital, Department of Laboratory Medicine, Singapore, Singapore.
authors.forename: G J Y
authors.initials: GJ
authors.lastname: Ngan
authors.forename: L M
authors.initials: LM
authors.lastname: Ng
authors.forename: R
authors.initials: R
authors.lastname: Jureen
authors.forename: R T P
authors.initials: RT
authors.lastname: Lin
authors.forename: J W P
authors.initials: JW
authors.lastname: Teo
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods*; D021903:DNA, Ribosomal Spacer / Q000235:genetics*; D006801:Humans; D009161:Mycobacterium / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D009164:Mycobacterium Infections / Q000175:diagnosis* / Q000382:microbiology; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012338:RNA, Ribosomal, 23S / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012846:Singapore
chemical_list: D021903:DNA, Ribosomal Spacer; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S